Enterococcus faecalis is a common resident of human intestinal flora and a frequent cause of infection in humans. Enterococci have grown in prominence over the past few decades due to their ability to cause fatal infections in hospitalized persons and their capacity to readily transfer virulence determinants, such as antibiotic resistances, to other microbes. The ability of this organism to persist in the environment, colonize a variety of hosts, and pass virulence capabilities to other pathogens makes elucidation of virulence mechanisms in this bacterium imperative. One such factor that has been recognized for its contribution to the virulence of E. faecalis is the toxin, cytolysin. Cytolysin is a novel toxin, capable of lysing a wide variety of cell types and organisms - both bacterial and eukaryotic. The cytolysin toxin consists of two subunits, CylLL and CylLs, both of which are extensively modified to generate their active forms. Two unique regulatory proteins, CylR1 and CylR2, are essential for controlling toxin expression. What makes this system unusual is that CylR1 and CylR2 bear no significant homology to known regulatory components in other bacterial systems, and function by an unknown mechanism to disable transcription of the cytolysin operon. Furthermore, one of the toxin subunits, CylLs, has been shown to induce cytolysin production. As cytolysin toxin is both a significant virulence factor of E. faecalis, and is subject to unique regulatory mechanisms, it is of interest to understand how this toxin is regulated in hopes of improving therapeutic measures to subvert infections by this organism. Therefore, this application aims to identify the mechanisms by which CylR1 and CylR2 regulate expression of cytolysin and to determine the role of CylLs in this regulatory process. To achieve these aims, we will examine the cellular localization of both CylR1 and CylR2, assess their ability to interact with each other and CylLs, and study the effects of these interactions on the regulation of the cytolysin operon. [unreadable] [unreadable] [unreadable]